The circadian clock organizes the physiology and behavior of organisms to their daily environmental rhythms. The central circadian timekeeping mechanism in eukaryotic cells is the transcriptional-translational feedback loop (TTFL). In theTTFL, the transcription factors CLOCK (CLK) and CYCLE (CYC) play crucial roles in activating expression of core clock genes and clock-controlled genes. Many signaling pathways converge on the CLK/CYC complex and regulate its activity to fine-tune the cellular oscillator to environmental time cues. We aimed to identify factors that regulate CLK by performing tandem affinity purification combined with mass spectrometry usingS2 cells that stably express HA/FLAG-tagged CLK and V5-tagged CYC. We identified SNF4Aγ, a homolog of mammalian AMP-activated protein kinase γ (AMPKγ), as a factor that copurified with HA/FLAG-tagged CLK. The AMPK holoenzyme composed of a catalytic subunit AMPKα and two regulatory subunits, AMPKβ and AMPKγ, directly phosphorylated purified CLKLocomotor behavior analysis inrevealed that knockdown of each AMPK subunit in pacemaker neurons induced arrhythmicity and long periods. Knockdown of AMPKβ reduced CLK levels in pacemaker neurons, and thereby reduced pre-mRNA and protein levels of CLK downstream core clock genes, such asandFinally, overexpression of CLK reversed the long-period phenotype that resulted from AMPKβ knockdown. Thus, we conclude that AMPK, a central regulator of cellular energy metabolism, regulates thecircadian clock by stabilizing CLK and activating CLK/CYC-dependent transcription.Regulation of the circadian transcription factors CLK and CYC is fundamental to synchronize the core clock with environmental changes. Here, we show that the AMPKγ subunit of AMPK, a central regulator of cellular metabolism, copurifies with the CLK/CYC complex inS2 cells. Furthermore, the AMPK holoenzyme directly phosphorylates CLKThis study demonstrates that AMPK activity regulates the core clock inby activating CLK, which enhances circadian transcription. In mammals, AMPK affects the core clock by downregulating circadian repressor proteins. It is intriguing to note that AMPK activity is required for core clock regulation through circadian transcription enhancement, whereas the target of AMPK action is different inand mammals (positive vs negative element, respectively). Drosophila Drosophila in vitro Drosophila period vrille Drosophila Drosophila in vitro Drosophila Drosophila SIGNIFICANCE STATEMENT
